Transcription Factor AcMYB5 Activates Flavonoid Biosynthesis and Enhances Resistance of Kiwifruit to Bacterial Canker

Abstract

Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), poses a serious threat to the global kiwifruit industry. Although flavonoids are widely recognized as natural antibacterial compounds, the transcriptional regulatory networks controlling their synthesis in kiwifruit and their relationship with production of downstream antibacterial metabolites remain poorly understood. In this study, we identified the transcription factor AcMYB5 as a key mediator of salicylic acid (SA) signaling that activates flavonoid biosynthesis and enhances resistance to Psa. Comparative analysis between the resistant cultivar ‘Jinkui’ and the susceptible cultivar ‘Hongyang’ revealed that Psa infection induced a rapid accumulation of endogenous SA, accompanied by a decrease in jasmonic acid (JA) levels in ‘Jinkui’. From a pool of SA-induced candidate genes, we identified AcMYB5, which is rapidly up-regulated by SA and encodes a nuclear localization protein. Overexpression of AcMYB5 in susceptible kiwifruit significantly enhanced resistance to Psa. Mechanistically, AcMYB5 directly binds to and activates the promoter of the chalcone isomerase (AcCHI), a key structural gene in the flavonoid pathway, leading to a marked increase in total flavonoid content. Notably, AcMYB5 did not activate any other genes in the flavonoid synthesis pathway in our assays, underscoring its target specificity. Our findings reveals a novel AcMYB5-AcCHI module that finely tunes flavonoid-mediated defense responses, offering valuable genetic targets and strategic insights for kiwifruit-resistant breeding.

1. Introduction

Kiwifruit (Actinidia spp.) is an economically important fruit crop, with global cultivation area and yield continuing to grow. The fruit is rich in vitamin C and is a variety of antioxidant [1]. However, bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) seriously threatens the sustainable development of the global kiwifruit industry [2]. Psa can infect stems, leaves and flowers, leading to plant wilting, cankers and even death, resulting in huge economic losses [3]. Therefore, excavating the inherent disease-resistant genetic resources of kiwifruit and elucidating its molecular mechanism of disease resistance are the basis for cultivating new disease-resistant varieties and developing green prevention and control strategies.

In recent years, it has been found that Psa strains are divided into five types: the first type is called Psa-J, which is susceptible to Hayward varieties and contains a gene cluster encoding bean toxin [4]; the second category is referred to as Psa-K, Hort16A- and Hayward-susceptible, containing the coding coronatine gene cluster [5]; the third category is referred to as Psa-V, which lacks the coding gene of bean toxin and coronatine gene, but has the most pathogenicity [6], causes the most serious harm and is susceptible to Chinese yellow meat kiwifruit and delicious green meat kiwifruit; the fourth type of Psa-LV, lacking part of the effector gene cluster, is less pathogenic; the fifth type of multiple sequence site analysis showed that it was similar to Psa-J, but there was no bean toxin gene [7]. There are obvious differences in the treatment of different genotypes of strains. The pathogenic mechanism of Psa leading to its infection of kiwifruit plants in the late outbreak is extremely rapid. When the toxic substances secreted by the pathogens accumulate to a certain amount in the plant, it will lead to the reduction in some biosynthesis metabolites related to growth and development, lesions on the surface of kiwifruit leaves.

Plants have developed a hierarchical immune system through long-term evolution in response to pathogen infection [8]. When attacked by pathogens, plants can activate defense responses by recognizing pathogen-associated molecular patterns (PAMP-Triggered Immunity, PTI) and effector-triggered immunity (ETI) [9,10]. The pathogenicity of Psa is multiple, and the direct transport of effector proteins (T3Es) to the cytoplasm of plant cells through the type III secretion system (T3SS) has the highest infection efficiency [11]. The synergistic effect of these effector proteins can not only inhibit PTI and mitogen-activated protein kinase cascade (MAPK cascade), but also affect the accumulation of callose and the synthesis of reactive oxygen species in the host [12]. In addition, Psa can also produce phytotoxins, of which crown toxin is particularly critical—it plays a role by mimicking the molecular structure of the plant hormone jasmonic acid-isoleucine (JA-Ile) [13]. By activating the jasmonic acid (JA) signaling pathway, crown toxin can not only inhibit the salicylic acid (SA)-mediated defense response, but also promote the reopening of stomata, thus creating a key invasion channel for bacteria. In plants, SA and JA signaling pathways play a central role. Recent studies have shown that a variety of plants have spectral resistance to different types of pathogens by using SA and JA dual-responsive promoters to drive the expression of antimicrobial peptides [14]. In rice, bacterial pathogens regulate the rice miR156-OsSPL7/14/17-OsAOS2/OsNPR1 regulatory network to attenuate SA- and JA-mediated plant defense and promote its infection [15]. In kiwifruit, AcC3H1 and AcREM14 enhanced the resistance to bacterial canker by activating the SA signaling pathway [16]. VvWRKY5 in grape enhances the resistance of grape to white rot by promoting the JA pathway [17]. They usually have antagonistic effects to coordinate the defense against different types of pathogens (such as living vegetative and dead vegetative) [18]. AcJAZ2L2 protein interacts with AcMYC2 in kiwifruit to inhibit the expression of JA-responsive genes, thereby enhancing the disease resistance of kiwifruit canker [19]. Studies have shown that the SA signaling pathway plays a leading role in resistance to living vegetative pathogens (such as Psa).

In addition to classical hormone signaling, plant secondary metabolites constitute another key component of the plant defense system [20]. Flavonoids, a class of polyphenolic compounds widely distributed in the plant kingdom [21], have been shown to possess direct antibacterial activity and contribute to disease resistance by strengthening cell walls and acting as antioxidants [22,23]. The biosynthesis of flavonoid derivatives was synergistically activated by CiNFYA1-CiGBF3-CiCHS2 transcription module in citrus, which significantly enhanced abiotic stress tolerance [24]. The biosynthesis of flavonoids is strictly controlled by a series of structural genes, with the MYB transcription factor family being considered the most critical regulatory switch in this pathway [25]. MsMYB206-MYB450 promotes the salt tolerance of alfalfa by regulating the biosynthesis and synthesis of flavonoids [26]. In tea, CsMYB67 precisely regulates the synthesis of flavonoids by delicately controlling the expression of CsFLS and CsANS [27]. However, for kiwifruit, it remains unclear whether and how specific MYB transcription factors integrate SA signals to regulate flavonoid metabolism, thereby conferring resistance to Psa.

In this study, the resistant cultivar ‘Jinkui’ and the susceptible cultivar ‘Hongyang’ were used as materials to preliminarily reveal the key role of SA/JA hormone balance in kiwifruit–Psa interaction. On this basis, we screened and identified an MYB transcription factor, AcMYB5, which is strongly induced by SA. Through genetic transformation, physiological, biochemical and molecular biology experiments, we systematically demonstrated that AcMYB5 promotes flavonoid accumulation by directly activating the expression of key genes for flavonoid synthesis, thereby significantly enhancing kiwifruit resistance to canker disease. This work not only decodes the function of an important disease resistance gene, but also provides a potential strategy and candidate target for the cultivation of new kiwifruit varieties with high resistance by regulating flavonoid metabolism.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Plant materials including kiwifruit (Actinidia chinensis), which is susceptible to cultivar ‘Hongyang’, resistant cultivar ‘Jinkui’ and seedlings of Nicotiana benthamiana (N. benthamiana) were included in this study. ‘Hongyang’ and ‘Jinkui’ plantlets were primarily used for Psa infection assays and hormonal assays. ‘Hongyang’ was also used as transcriptome analysis and the background for generating transgenic lines. All kiwifruit materials were maintained and propagated under tissue culture conditions on MS medium at 25 °C with a 16 h light/8 h dark photoperiod. N. benthamiana plants were grown in a growth chamber under the same conditions for transient transformation assays. The Psa bacterial strain JF8 [28] was successfully transferred the GFPuv label as described by Gao et al. [29] was used for all infection assays. The plant materials used in this study were sourced from the germplasm resource repository of the Key Laboratory of Horticultural Crop Quality Biology in Anhui Province.

2.2. In Vitro Infection Assay and Bacterial Growth Counting

Psa was cultured overnight, harvested and resuspended in 10 mM MgCl₂ buffer to an optical density at 600 (OD₆₀₀) nm of 0.1. For the leaf disk assay, circular disks (1 cm diameter) were excised using a sterile puncher. The disks were immersed in the bacterial suspension for 5 min and then subjected to vacuum infiltration at 0.06 MPa for 2 min, repeated three times. After infiltration, excess liquid was removed using sterile filter paper, and the disks were transferred onto solid MS medium. For the seedling immersion assay, seedlings at the same developmental stage were immersed in the bacterial suspension (OD₆₀₀ = 0.1) for 5 min. Residual inoculum was removed with sterile filter paper, and the seedlings were transferred to fresh MS solid medium for further growth. The observation of inoculation phenotype needs about three weeks.

All inoculated materials were incubated at 16 °C and 75% relative humidity for 5 days. After incubation, disease phenotypes were recorded. To determine bacterial populations, leaf disks were rinsed with sterile water, blotted dry and weighed (1.0 g per sample). The tissues were ground in 2 mL of sterile water using a mortar and pestle, and serial dilutions (10⁰ to 10⁻⁴) were prepared. Fifty microliters from each dilution were plated onto solid medium, with three replicates per dilution. Plates were incubated at 28 °C for 36 h, after which colony-forming units (CFUs) were counted for statistical analysis.

2.3. Kiwifruit Stable Transformation

The coding sequence (CDS) of AcMYB5 was cloned into a pCAMBIA1300-derived vector by the CaMV35S promoter. The recombinant plasmid was introduced into Agrobacterium tumefaciens strain EHA105 (Weidi Biotechnology, Shanghai, China), which was then used to transform leaf explants of the ‘Hongyang’. The transformed calli were selected on hygromycin-containing medium (Coolaber, Beijing, China) and regenerated into shoots. Positive transgenic plantlets (AcMYB5-OE) were confirmed by RT-qPCR. Wild-type (WT) HY plantlets were used as controls. The primers are listed in Supplementary Table S1.

2.4. RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

The previously described protocol was used for the extraction of total RNA from various kiwifruit samples, including SA-treated leaves (‘Jinkui’), WT (‘Hongyang’) and transgenic plant leaves (AcMYB5-OE), using a Plant RNA Extraction Kit (azyme, Nanjing, China). Their reverse transcription and real-time quantitative PCR were consistent with [30]. The GAPDH gene was used as an internal control, and each sample was subjected to 3 independent replicates. Relative gene expression was calculated according to the 2^−ΔΔCt method [31]. The primers used are listed in Supplementary Table S1.

2.5. Dual-Luciferase Assay

The full-length coding sequences of AcMYB5 was cloned into pGreenII-62-SK vector to serve as effectors. The CAACC motifs in DNA fragments of AcCHS, AcCHI, AcF3H, AcDFR, AcFLS and AcFNSII were cloned and inserted upstream of the firefly luciferase (LUC) gene in pGreenII-0800-LUC to generate the reporters. The effector and reporter were introduced into Agrobacterium strain GV3101 (Weidi Biotechnology, Shanghai, China), and were co-transformed into leaves of 4-week-old N. benthamiana in ratios of 1:1 (effector/reporter) following an established protocol. The transformation of the pGreenII-62-SK empty vector with either reporter (AcCHS, AcCHI, AcF3H, AcDFR, AcFLS and AcFNSII) was used as a negative control. LUC activity was measured using a dual-LUC reporter assay system (Beyotime Biotechnology, Shanghai, China). Three biological replicates were conducted. The relative LUC activity was indicated as the ratio of firefly LUC signal to renillaREN signal. Primers are listed in Table S1.

2.6. SA and JA Quantification

For hormone quantification (Figure 1), leaf samples from ‘Jinkui’ and ‘Hongyang’ were collected at 0 and 12 h with Psa or mock treatment (10 mM MgCl₂). The samples were immediately frozen in liquid nitrogen and stored at −80 °C. The frozen tissue was ground to a fine powder, and SA and JA were extracted and quantified using a commercial SA and JA content detection kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. The hormone content was measured spectrophotometrically and expressed as ng per gram of fresh weight.

2.7. Determination of Flavonoid Content

The flavonoid content in the leaves of wild-type (WT) and AcMYB5-OE transgenic kiwifruit lines was determined using a plant flavonoid content detection kit (Solarbio, Beijing, China). according to the manufacturer’s instructions. The hormone content was measured spectrophotometrically and expressed as mg per gram of fresh weight.

2.8. Statistical Analysis

The data are presented as the mean of at least three independent experiments. The data were statistically processed using the GraphPad Prism version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA), and the statistical significance of the differences between the groups was evaluated using analysis of variance (ANOVA). The significance levels were set at p < 0.05 (*), p < 0.01 (**) p < 0.001 (***), p < 0.0001 (****) and p > 0.05 (ns, not significant).

3. Results

3.1. Hormone Signal Changes in Resistant and Susceptible Varieties After Infection with Psa

To explore the different defense responses of kiwifruit to Psa, we selected the resistant variety ‘Jinkui’ (JK) and the susceptible variety ‘Hongyang’ (HY) for the Psa immersion inoculation assay (Supplementary Figure S1). Compared with ‘HY’, ‘JK’ showed significantly milder disease location and severity (Figure 1A). Bacterial growth experiments confirmed that the bacterial population in ‘JK’ was significantly lower than that in ‘HY’ (Figure 1B), which was consistent with phenotypic observation. We further monitored the dynamic changes in defense hormones and found a contrasting pattern between the two varieties. In ‘JK’, Psa infection triggered a rapid increase in SA levels at 12 h after inoculation, accompanied by a decrease in JA. On the contrary, the susceptible cultivar ‘HY’ showed a decrease in SA and a simultaneous increase in JA (Figure 1C,D). These results demonstrated that the SA/JA hormone balance was broken in the early stage of infection, and the accelerated accumulation of SA and the inhibition of JA were essential for the establishment of effective defense.

Figure 1. Comparative analysis of defense responses in kiwifruit varieties after infection with Psa. (A) Observation of lesion size on the leaves of the resistant cultivar ‘Jinkui’ (‘JK’) and the susceptible cultivar ‘Hongyang’ (‘HY’) kiwifruit after 20 days of Psa infection. The leaves were taken from typical disease symptoms in different biological replicate samples. Scale bar = 1 cm. (B) Bacterial counting of the growth of Psa from ‘JK’ and ‘HY’ kiwifruit leaves after 5 days of Psa infection. (C) Endogenous SA contents of leaves of ‘JK’ and ‘HY’. (D) Endogenous JA contents of leaves of ‘JK’ and ‘HY’. The data represent mean ± SE (n = 3). Asterisks indicate that the value is significantly different from that of the ‘HY’ and 0 h, where p < 0.01 (**) p < 0.001 (***), p < 0.0001 (****), and p > 0.05 (ns, not significant).

3.2. Screening of Genes Responding to Salicylic Acid Induction

To explore the regulatory role of SA in kiwifruit disease resistance, based on the transcriptome analysis of the ‘JK’ after inoculation with Psa, we screened 27 differentially expressed genes (12 up-regulated and 15 down-regulated) [30]. Promoter cis-acting element analysis of these gene promoters showed that 12 genes contained SA response elements. To verify whether these candidate genes were induced by SA, we detected the gene expression dynamics of ‘JK’ at three time points under exogenous SA treatment by RT-qPCR (Figure 2). The results showed that not all genes containing SA elements responded to SA treatment, and the expression patterns of each response gene were significantly time specific. Most genes, including Acc12804, Acc22332, Acc17393 and Acc30931, exhibited a transient induction pattern, and their expression levels began to decline after being induced to the highest point by SA. It is worth noting that the induction of Acc22332 was the most significant, and the expression level was up-regulated by about 10-fold after 12 h of SA treatment. In contrast, some genes such as Acc18803 and Acc22908 showed delayed response characteristics. The expression of the former was significantly increased by 3.5 times at 24 h, while the expression of the latter was significantly inhibited by 80%. The remaining genes (Acc32296, Acc07632, Acc08521) showed only weak induction, while Acc19875, Acc03329 and Acc29999 had no significant response to SA treatment. Given that Acc22332 showed the fastest and strongest transcriptional activation of SA signal, we selected it as the core candidate gene for subsequent functional analysis.

Figure 2. Time-course expression patterns of 12 candidate genes in the resistant cultivar ‘Jinkui’ after SA treatment ((A)–(L)). Time-course expression pattern 12 candidate genes in the resistant cultivar ‘Jinkui’ after SA treatment. Gene expression was analyzed by RT-qPCR at indicated time points (hpi). The data represent mean ± SE (n = 3). Asterisks indicate that the value is significantly different from that of the 0 h, where p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p > 0.05 (ns, not significant).

3.3. Overexpression of AcMYB5 Enhances Kiwifruit Resistance to Psa

Phylogenetic analysis showed that Acc22332 protein was highly homologous to Arabidopsis MYB5 gene, so it was named AcMYB5 in this study (Supplementary Figure S2). Subcellular localization results showed that AcMYB5 is a nuclear protein (Supplementary Figure S3). To verify the function of AcMYB5, we obtained AcMYB5 overexpressing (AcMYB15-OE) lines by genetic transformation (Figure 3A). RT-qPCR showed that the expression levels of AcMYB5 were three to nine times higher in the OE lines than in the wild-type (WT) plants (Figure 3B). To assess the resistance of transgenic AcMYB5 to Psa, transgenic leaves were infected with Psa. At 5 days, AcMYB15-OE leaves showed smaller lesions compared with WT (Figure 3C). Meanwhile, bacterial growth assays showed a significant decrease in the bacterial population of Psa in AcMYB5-OE (Figure 3D). These results indicate that AcMYB5 can enhance the resistance of kiwifruit to bacterial canker by inhibiting the proliferation of Psa pathogens.

Figure 3. Expression and functional analysis of AcMYB15. (A) Observations of AcMYB5-OE and wild type of ‘Hongyang’. Scale bar = 3 cm. (B) Expression profiles of AcMYB5 in AcMYB5-OE. (C) Observation of adaxial leaves lesions in the WT and AcMYB15-OE 5 d after Psa infection. Scale bar = 0.5 cm. (D) Bacterial counting of the growth of Psa from WT and AcMYB5-OE leaves. Data represent means ± SE (n = 3). Asterisks indicate that the value is significantly different from that of the WT, where p < 0.01 (**), p < 0.0001 (****), and p > 0.05 (ns, not significant).

3.4. AcMYB5 Transcriptionally Activates AcCHI to Promote Flavonoid Synthesis

Previous studies indicated a positive correlation between MYB5 expression and multiple flavonoid biosynthetic genes. To clarify its mechanism, we measured the content of flavonoids in AcMYB5 transgenic kiwifruit and found that its content was significantly increased. In addition, six key genes involved in flavonoid biosynthesis were detected in AcMYB5 seedlings. Compared with the wild-type, AcCHI and AcFLS were significantly up-regulated in the AcMYB5-OE strain, while AcCHS, AcF3H, AcDFR and AcFNSII did not change significantly. To determine whether AcMYB5 directly regulates these target genes, we performed a dual-luciferase (Dual-LUC) reporter assay using the promoters of six key flavonoid biosynthetic genes. The results showed that AcMYB5 significantly enhanced the promoter activity of AcCHI, with an activation level approximately 3-fold-higher. In summary, our results demonstrate that AcMYB5 acts as a key regulator of flavonoid biosynthesis, specifically by directly activating the AcCHI promoter and potentially indirectly upregulating AcFLS expression. This regulatory role leads to a significant accumulation of flavonoids, thereby elucidating a key part of the molecular mechanism by which AcMYB5 enhances disease resistance.

4. Discussion

Bacterial disease is a key biotic stress factor restricting the sustainable development of industry. Cultivating disease-resistant varieties is the fundamental way to achieve green prevention and control, and its success depends on the in-depth analysis of plant immune mechanisms, especially hormone signaling pathways. As a key defensive metabolite, flavonoids can not only directly inhibit pathogens, but also regulate plant immune responses. However, the transcriptional regulation mechanism of flavonoid accumulation in kiwifruit under pathogen infection is still unclear. This study identified that the kiwifruit transcription factor AcMYB5 plays a central role in resistance to bacterial canker. In the resistant cultivar ‘Jinkui’, pathogen infection triggered the accumulation of endogenous SA, which in turn induced the expression of AcMYB5. Functional studies have shown that AcMYB5-OE significantly enhances resistance to canker disease, and its mechanism is to directly activate the key gene AcCHI in the flavonoid synthesis pathway, thereby driving flavonoid accumulation. This study revealed a new defense pathway composed of SA signal-AcMYB5-flavonoid metabolism.

Bacterial canker, a devastating disease caused by Psa, has posed a continuous threat to the global kiwifruit industry [32]. In the plant immune system, SA, as a key signaling molecule, plays a central role in the establishment of systemic acquired resistance, and can activate the expression of a series of downstream defense-related genes [33]. Plant hormones SA and JA have complex interrelationships in plant defense, mainly manifested as antagonistic and synergistic modes. In poplar, SA and JA are synergistically accumulated to activate the expression of defense genes and enhance the resistance of plants to rust [34]. Under biotic stress, it is more antagonistic. SA is mainly involved in the defense of plants against living vegetative pathogens. JA mainly regulates dead vegetative pathogens and insect invasion. Both of them provide an important molecular basis for plants to maintain the balance between growth and defense in complex environments through hormone receptors and signal transduction [35]. In our previous study, we observed that Psa infection rapidly induces the accumulation of endogenous SA in the disease-resistant cultivar ‘JK’ [30], which was highly consistent with the classical function of SA in resistance to pathogens. The conduction of SA signal depends on the cascade activation of its downstream transcriptional regulatory network [36]. This study found that AcMYB5 is a key regulatory node in this signaling pathway, and its promoter region contains SA response elements, and its expression is rapidly and strongly induced under SA treatment, indicating that AcMYB5 is an important part of the early response process of SA, and may act as a ‘molecular switch’ to convert transient hormone signals into continuous transcriptional activation. In model plants such as Arabidopsis and rice, multiple MYB transcription factors have been reported to be involved in SA-mediated immune responses. For example, AtMYB96 regulates cuticular wax synthesis by integrating SA and abscisic acid signaling pathways to enhance plant disease resistance [37]. OsMYB30 improved rice insect resistance by increasing SA and lignin accumulation [38]. This study showed that the conserved disease resistance module of MYB-SA was still established in kiwifruit, and functional specificity was generated on this basis. By precisely regulating its specific downstream target gene network, AcMYB5 endowed kiwifruit with effective resistance to Psa, revealing the evolutionary mechanism of the regulatory module being ‘customized‘ under specific phylogenetic background and pathogen stress.

This study reveals a mechanism: AcMYB5 exhibits a highly specific regulation of the flavonoid synthesis pathway, rather than a regulatory master switch. Assays have confirmed that it can directly bind to and activate the AcCHI promoter, but has no significant direct regulatory effect on key genes such as AcCHS and AcF3H. This finding, together with recent studies, reveals the precise regulation of MYB transcription factors. For example, in tomato, SlMYB75 has been shown to achieve branch-specific regulation of phenylpropanoid metabolic pathways by specifically recognizing different cis-elements [39]. This mechanism has been confirmed in apples, and MdMYB9 specifically activates proanthocyanidin synthesis genes after interacting with specific bHLH proteins [40]. This specific binding suggests that AcMYB5 may achieve more precise regulation of flavonoid synthesis by forming a specific MBW ternary complex [41]. AcMYB5 effectively promotes flavonoid synthesis by specifically enhancing its expression, avoiding the break of growth–defense balance. The precise regulation exhibited by AcMYB5 is consistent with the study of VvMYB5b in grapes, which avoids metabolic imbalance by accurately regulating specific branch pathways [42]. In addition, in AcMYB5-OE plants, the level of endogenous SA was higher than that of WT (Supplementary Figure S4A), indicating that there may be a synergistic relationship between the flavonoid metabolic pathway and the salicylic acid pathway. In plant defense response, flavonoids and SA are often involved in disease resistance and stress resistance. SA is the core hormone of plant immunity, and flavonoids can clear reactive oxygen species, activate plant immune signaling pathways as signal molecules and indirectly induce the synthesis of SA, activating the immune response [43]. At the same time, the flavonoid synthesis pathway and the SA synthesis pathway share some intermediate metabolites (phenylalanine, cinnamic acid). The accumulation of flavonoids may indirectly promote the synthesis of SA by regulating the fate of such metabolites [44]. However, it was found that there was no significant change in the content of endogenous JA, indicating that the specific regulation of transcription factors finely regulated SA-flavonoids without affecting the JA-mediated regulatory network. We obtained several independent AcMYB5-OE lines in this study (Figure 3A,B). This variation is likely to be tissue-specific and copy number differences in transgenic plants. However, it is important that although there are differences in transcript abundance, it is different from the wild-type (Figure 3C,D and Figure 4A), all AcMYB5-OE lines showed enhanced resistance to Psa and increased flavonoid accumulation. This clear correlation between AcMYB5 expression and disease-resistant phenotype strongly supports our conclusion. In summary, this study systematically elucidated the complete disease resistance pathway of SA signaling in fruit trees initiated by the pathogen Psa, conducted by AcMYB5, and finally driven by specific activation of AcCHI to drive flavonoid synthesis. This finding not only deepens the understanding of the immune mechanism of fruit trees, but also provides theoretical support and genetic resources for kiwifruit molecular breeding, which is of great value to promote the green development of the industry.

Figure 4. AcMYB5 activates the expression of AcCHI. (A) Total flavonoid content from WT and AcMYB5-OE leaves. ((B)–(G)) The expression level of important genes for flavonoid synthesis in WT and AcMYB5-OE by RT-PCR. (H) Schematic diagram of the vectors used for the dual-luciferase (LUC) assays. P35S, the CaMV35S promoter. LUC, firefly luciferase. REN, Renilla luciferase. (I) A representative bioluminescence image of tobacco leaf injected with different combinations of vectors. Data represent means ± SE (n = 3). Asterisks indicate that the value is significantly different from that of the WT and empty vectors, where p < 0.05 (*), p < 0.01 (**) p < 0.001 (***), p < 0.0001 (****) and p > 0.05 (ns, not significant).

5. Conclusions

This study found a defense pathway in kiwifruit that SA regulates flavonoid participation, which integrates salicylic acid signal transduction and flavonoid metabolism, thereby conferring kiwifruit resistance to bacterial canker disease. We found that the transcription factor AcMYB5 is a regulator of this pathway, which can directly and specifically activate the promoter of AcCHI, a key gene for flavonoid biosynthesis, under the rapid induction of salicylic acid. This targeted regulation mechanism effectively promotes the accumulation of defensive flavonoids, thereby achieving efficient defense.

It is worth noting that the SA-AcMYB5-flavonoid module finely regulates each hormone involved in disease resistance. A small increase in endogenous SA levels in AcMYB5-OE indicates that there is a synergistic effect between the flavonoid metabolic pathway and the SA pathway, which may lay a foundation for plants to initiate a stronger defense mechanism. At the same time, the phenomenon that JA levels remain unchanged indicates the fineness of the module—it effectively circumvents the usually antagonistic cross-regulation between SA and JA signaling pathways, which is essential for initiating appropriate defense against the semi-biological pathogen Psa.

Our research results not only clarify a new and accurate molecular mechanism in the immune mechanism of kiwifruit, but also provide valuable genetic resources and breeding targets for breeding work. The AcMYB5-AcCHI module has great potential in the field of molecular breeding and can be used to cultivate a new generation of kiwifruit varieties with enhanced and sustainable resistance, thus contributing to the green and sustainable development of the global kiwifruit industry.